Analogs of parathyroid hormone and pth-related protein as bone anabolic agents

ABSTRACT

The invention provides novel parathyroid hormone analogs and parathyroid hormones-related protein analogs. The invention also provides methods of using these analogs to treat osteoporosis, promote the formation of bone, and inhibit bone loss.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.provisional application 60/388,918, filed Jun. 13, 2002, and U.S.provisional application 60/398,005, filed Jul. 23, 2002, each of whichis incorporated herein by reference.

GOVERNMENT SUPPORT

This work was funded in part by Grant Number DK47940 from the NationalInstitutes of Health. Accordingly, the Government may have certainrights to this invention.

FIELD OF THE INVENTION

The invention relates to parathyroid hormone analogs and parathyroidhormone-related protein analogs and their use as bone anabolic agents.These analogs are useful for treating osteoporosis, promoting theformation of bone, and inhibiting bone loss.

BACKGROUND OF THE INVENTION

Osteoporosis is the most common form of metabolic bone disease. The termencompasses any disease or idiopathic cause that results in a reductionin the mass of bone per unit volume. Osteoporosis results in differingdegrees of skeletal fragility sufficient to increase the risk offracture. Typically, the resorption in bone mass results from animbalance in the processes that influence the acquisition andmaintenance of skeletal mass. Osteoporosis is the most common disorderin which all of the skeleton is. involved, and is an important cause ofmorbidity in the elderly. Post-menopausal women are at a particularlyhigh risk for idiopathic osteoporosis.

In general, cancellous bone has higher turnover rate than cortical bone.In early years of menopause the rate of bone loss is substantiallyfaster for vertebral cancellous bone than for cancellous bone at othersites and cortical bone. Usually, peak adult bone mass is reachedbetween the ages of 30 and 35 for cortical bone and often even earlierfor trabecular bone. After peak adult bone mass is reached, the rates ofbone formation and resorption are typically approximately equal, andrelatively low compared with the period of growth spurt. Normally,skeletal mass is maintained by the normal balance between bone formationand bone resorption.

Bone remodeling, including bone formation and resorption, is acontinuous process. In healthy subjects, these complementary mechanismsmaintain a steady bone mass per unit volume (referred to herein as “bonemass”). However, subjects that may have failed to obtain optimalskeletal mass during the first 30 years of life, and/or subjects whereinthe rate of bone resorption exceeded the rate of bone formation afterpeak skeletal mass was obtained may develop osteoporosis.

Bone resorption typically precedes formation, but does not last as longas formation. As a result, there are typically more sites of activeformation than of resorption. Unless formation compensates forresorption, the bone mass will decrease. After an age ranging from 40 to50, cortical bone is lost at a rate of about 0.3 to 0.5% per year inboth men and women. There is an accelerated loss of cortical bonesuperimposed on the age-related loss around the menopause in women. Thecumulative losses of bone mass range from 20 to 30% in men and up to 50%in women. Remodeling activity is typically increased in women withpost-menopausal osteoporosis, when compared with age-matched controls.The difference is even more striking when the bone remodeling activitiesof post-menopausal women are compared to pre-menopausal women. When thedifference between the rates of bone formation and bone resorption ismaintained, the loss of bone density may become so marked that the bonecan no longer withstand normal mechanical forces to which it issubjected, resulting in bone fracture. Often, osteoporosis is onlyrecognized as a clinical problem after a fracture has occurred.

In some instances, another disease, for example, Cushing's Syndrome orosteogenesis imperfecta, results in osteoporosis. In most cases,however, patients with osteoporosis do not appear to have any otherdisease. Osteoporosis, which occurs in children, young adults, andadults of both sexes with normal gonadal function, is referred to asidiopathic osteoporosis, although often the osteoporosis is of unknownpathogenesis.

There are agents available for preventing bone loss and treatingestablished osteoporosis. The ability to measure bone density andpredict bone fracture risk has changed the approach to treatment, as aclinical benefit of treatment can be measured in terms of improved bonemass as well as a decreased incidence of fractures. Most of the drugsthat are available are inhibitors of bone resorption, although someagents, such as sodium fluoride, increase bone formation. Fluoridetreatment suffers from a narrow therapeutic window and a lack ofdemonstration of reduction in fracture rate, probably due to the poormechanical qualities of newly formed bone. The agents that are currentlyavailable and/or under investigation to treat osteoporosis include, butare not limited to, Selective Estrogen-Receptor Modulators (SERMs),estrogens, androgens, cathepsin K inhibitors, calcium supplements,vitamin D (and metabolites and analogs thereof, thiazidediuretics,-calcitonin (and analogs thereof), bisphosphonates, fluoride,integrin antagonists, parathyroid hormone, calcilytics, calcimimetics,inhibitors of Src tyrosine kinase and Src SH2 inhibitors.

However, the available agents to treat osteoporosis that are availableto date suffer from several drawbacks. First, most available agents mayinhibit bone resorption but do not promote bone formation. Additionally,many women are reluctant to use estrogens to prevent bone loss and/ortreat osteoporosis because of the return of menstrual bleeding and thefear of an increased risk for endometrial and breast cancer.Testosterone has been shown to be useful to treat osteoporotic men withgonadal deficiency, but there has been no evidence of efficacy in menwith no normal gonadal function. Some patients cannot tolerate theagents used to treat osteoporosis because of undesirable side-effects,such as knee, foot and ankle pain, and nausea. Additionally, none of theagents used to treat osteoporosis completely inhibit resorbtion and/oractively promote bone formation.

As a result, several groups have been investigating various types ofagents to treat osteoporosis, alone, and in combination. Parathyroidhormone (PTH), parathyroid related-protein (PTHrP), and analogs thereofare among the many agents that have been proposed for the treatment ofosteoporosis.

The normal function of PTH is to maintain extracellular fluid calciumconcentration. PTH acts directly on bone and kidney and indirectly onthe intestines. PTH production in healthy individuals is closelyregulated by the concentration of serum ionized calcium. Tendenciestowards hypocalcemia, for example, induced by a calcium-deficient diet,are balanced by an increased PTH secretion. The increase in PTH levelsincreases the rate of bone resorption, thereby increasing the calciumflow from bone into blood, reduces the renal clearance of calcium. andincreases the efficiency of calcium absorption in the intestines.

The physiological role of the parathyroid hormone-related protein(PTHrP) is not fully understood, but is thought to be acting principallyas a paracrine or autocrine factor. PTHrP plays a role in fetaldevelopment as well as in adult physiology. PTHrP is produced by manycell types, including brain, pancreas, heart, lung, memory tissue,placenta, endothelial, and smooth muscle cells. In adults, PTHrP isthought to have little to do with calcium homeostasis, except in diseasestates.

PTH and PTHrP are distinct proteins and products of different genes.However, they share a similar bioactivity profile and a very limitedsequence homology, indicating that they may have evolved from a commonancestral gene. Eight out of the 13 first amino acid residues at theN-terminus are identical. Both PTH, an 84 amino acid residues peptide,and PTHrP, a 139 to 173 amino acid residues peptide, bind to the PTHreceptor (often referred to as the PTH/PTHrP receptor) and stimulate thesame intracellular signaling pathways.

The mature circulating form of parathyroid hormone is comprised of 84amino acid residues. For most bone-related activities the truncated formof PTH, PTH(1-34), is a full agonist like the native 84 amino-acidhormone. Amino-terminal truncation results in polypeptides that arecompetitive antagonists of PTH-stimulated adenylate cyclase. Forexample, [Tyr³⁴]bPTH(7-34)NH₂ retains moderate affinity for renal PTHreceptors, but does not have any agonist activity; weak receptor bindingactivity is retained in a fragment as small as PTH(25-34) (M.Rosenblatt, et al., Endocrinol., (1980)107, 545-550). In contrast,carboxyl-terminal truncations of PTH(1-34) produce agonists withprogressively lower affinities. PTH(1 -25) is reported to be essentiallyinactive (Segre, G. V., et. al., J. Biol. Chem. (1979) 254, 6980-6996;Rosenblatt, M., Endocrinology of Calcium Metabolism; Parsons, J. A., ed.(1982) Ravens Press, N.Y., 103-142; Tregear, et al., Endocrinol. (1973)93, 1349-1353). The principal receptor-binding domain of PTH is reportedto include amino acid residues 25-34 and the principal activation domainis reported to include amino acid residues 1-6.

Under some circumstances, PTH is a bone anabolic agent, and promotesbone formation. However, PTH can stimulate bone resorption as well. Ithas been reported that high-dose, continuous administration of PTHresults in a lowered bone mass but low-dose, intermittent administrationof PTH can increase bone mass. PTH administered continuously reportedlycauses an increase in the number of bone cells, including osteoclasts,and an increase in bone remodeling. These increases reportedly areapparent within hours after PTH administration and persist for hoursafter PTH is withdrawn. PTH administration intermittently over days inhumans and animals reportedly leads to a net stimulation of boneformation. For example, see Neer et al., N. Engl. J Med. 344:1434-1441,(2001). In contrast, continuous exposure to elevated levels of PTH leadsto osteoclast-mediated bone resorption. Several groups have investigatedthe use of PTH and PTHrP analogs as agents to treat osteoporosis. Theseefforts are described in U.S. Pat. No. 5,747,456; U.S. Pat. No.5,849,695; U.S. Pat. No. 4,656,250; U.S. Pat. No. 6,051,686; and U.S.Pat. No. 6,316,410.

A need exists for pharmaceutical agents that can be used to treatosteoporosis. Preferably, such agents will have improved functionalactivity with minimum adverse side effects.

SUMMARY OF THE INVENTION

The present invention relates to polypeptide analogs which have agonistproperties similar to parathyroid hormone or parathyroid hormone-relatedprotein. These polypeptide analogs are useful in the treatment ofpatients with bone loss. Bone loss may result from conditions such asosteoporosis, glucocorticoid-induced bone loss, hypercortisolism (bothsubclinical and clinical), cancer, hypercalcemia, renal failure or otherkidney disorders, renal transplant and accompanying pharmacologicaltreatments, cholestatic liver diseases, viral hepatitis, bone losscaused by liver transplant, hyperparathyroid disease, bronchial asthma(including hormone-dependent), disorders due to haemodialysis, andosteomalacia. Often bone loss is attributed to idiopathic osteoporosis.

In some aspects of the invention, analogs of parathyroid hormone (PTH)and parathyroid hormone-related protein (PTHrP) are provided. Theanalogs include shortened forms of human or bovine PTH or human PTHrPpolypeptides comprising one or more amino acid substitutions orpseudopeptide units. The amino acid substitutions and pseudopeptideunits useful for preparing PTH and/or PTHrP analogs are described ingreater detail herein.

According to one aspect of the invention, analogs of PTH or PTHrP thatinclude one or more amino acid substitutions at positions 16, 17, 18, 19and /or 20 are provided. In preferred embodiments, the amino acidsubstitutions are β³-residues (β-substituted β-amino acid residues)although β² -residue substitutions (α-substituted β-amino acid residues)also are provided. In certain preferred embodiments, the analogs includeone or more amino acid substitutions selected from the group consistingof:

(a) at position 16: ,β-hAsn, β-hSer, β-hGln, β-hThr, β-hAla, β-Ala,β-hRaa, wherein β-hRaa=NH—CH(R)CH₂CO, where R=Et, nPr, (CH₂)_(n)CONH₂(n=0, 3 and 4);

(b) at position 17: β-hAsp, β-hSer, β-hGlu, β-hThr, β-hAla, , β-Ala,β-hRaa, wherein β-hRaa=NH—CH(R)CH₂CO, where R=Et, nPr, (CH₂)_(n)CO₂H(n=0, 3 and 4);

(c) at position 18: β-hMet, βhNle, β-hLeu, β-hIle, β-Val, β-hAla, β-Ala,β-hRaa, wherein β-hRaa=NH—CH(R)CH₂CO, where R=Et, nPr;

(d) at position 19: β-hAsp, β-hSer, β-hGlu, β-hThr, β-hArg, β-hLys,β-hDap, β-hDbu, β-hOrn, β-hAla, β-Ala, β-hRaa, wherein(β-hRaa=NH—CH(R)CH₂CO, where R=(CH₂)_(n)Guanidino or guanidine groupsurrogates (n=1, 2 and 4), (CH₂)_(n)CO₂H (n=0, 3 and 4); and

(e) at position 20: β-hArg, β-hLys, β-hDap, β-hOrn, β-hAla, β-Ala,β-hRaa, wherein β-hRaa=NH—CH(R)CH₂CO where R=(CH₂)_(n)Guanidino orguanidine group surrogates (n=1, 2 and 4).

Pharmaceutically acceptable salts of the foregoing analogs also areprovided.

In certain embodiments, the analogs or pharmaceutically acceptable saltsthereof also include one or more of the following amino acidsubstitutions: Glu at position 22; Leu or 2-Nal at position 23; Glu atposition 25; Lys or Aib at position 26; Leu at position 27; Leu atposition 28; Glu at position 29; Lys or Leu at position 30; Leu atposition 31; Thi at position 32; and Tyr at position 34.

In some preferred embodiments of the foregoing analogs orpharmaceutically acceptable salts thereof, the one or more amino acidsubstitutions are selected from the group consisting of (β-Ala atposition 16, β-Ala or β-hSer at position 17, β-Ala or β-hLeu at position18, β-Ala or β-hGlu at position 19, and (β-Ala at position 20.

In still other preferred embodiments of the foregoing analogs orpharmaceutically acceptable salts thereof, Nle is substituted for theMet at position 18 in those analogs where there is not a β amino acid atposition 18.

In particularly preferred embodiments, the analogs or pharmaceuticallyacceptable salts thereof include an amino acid sequence set forth as anyof SEQ ID NOs:13-41.

Some of the analogs or pharmaceutically acceptable salts thereofprovided according to the invention also include a pseudopeptide unit.The pseudopeptide unit preferably is a pseudodipeptide unit comprisingX_(xx) ¹Ψ(CH₂NH)Yyy², wherein Xxx is an amino acid selected from thegroup consisting of Ala, Gly, Thr and Ser, and Yyy is an amino acidselected from-the group consisting of Val, Leu, Nle, Ile and Phe.

According to a second aspect of the invention, analogs of PTH or PTHrPor pharmaceutically acceptable salts thereof that include an amino acidsubstitution at position 12 and a pseudopeptide unit are provided. Theamino acid substituted at position 12 preferably is 2-aminoisobutyricacid (Aib). The pseudopeptide unit preferably is a pseudodipeptide unitcomprising Xxx¹Ψ(CH₂NH)Yyy², wherein Xxx is an amino acid selected fromthe group consisting of Ala, Gly, Thr and Ser, and Yyy is an amino acidselected from the group consisting of Val, Leu, NMe, Ile and Phe.

In some embodiments of the second aspect of the invention, the analog orpharmaceutically acceptable salt thereof also includes one or more ofthe following amino acid substitutions: Nle at position 8; Nle atposition 18; 2-Nal at position 23; and Tyr at position 34.

In preferred embodiments of the second aspect of the invention, theanalog or pharmaceutically acceptable salt thereof includes the aminoacid sequence set forth as any of SEQ ID NOs:4, 42 or 43.

According to a third aspect of the invention, analogs of PTH or PTHrP orpharmaceutically acceptable salts thereof are provided that include aset of amino acid substitutions selected from the group consisting of

set (a): Glu at positions 25 and 29, and Lys 26 and/or 30;

set (b): Leu at positions 23 and 31, and Leu at position 27 or position28; and

set (c): Aib at position 26.

For certain analogs having set (a) amino acid substitutions, the analogsalso include one or more of the following amino acid substitutions: Gluat position 22, Leu at positions 23, 27, 28, 30 and 31, and Thi atposition 32. In preferred embodiments, the analogs include the aminoacid sequence set forth as any of SEQ ID NOs:5, 6, 9, 10 or 12.

For certain analogs having set (b) amino acid substitutions, the analogsalso include the amino acid sequence set forth as SEQ ID NO:7 or SEQ IDNO: 1 1.

For certain analogs having set (c) amino acid substitutions, the analogsalso include the amino acid sequence set forth as SEQ ED NO:8.

Any of the foregoing analogs or pharmaceutically acceptable saltsthereof can include two or more amino acid substitutions, three or moreamino acid substitutions, or four or more amino acid substitutions.

Any of the foregoing analogs or pharmaceutically acceptable saltsthereof also can include an amino acid substitution at position 5.Preferably the amino acid substitution at position 5 is His or Ile.

Any of the foregoing analogs or pharmaceutically acceptable saltsthereof also can have a modified carboxy terminus. Preferred carboxyterminus modifications include amides or alkylamides.

In preferred embodiments of the foregoing analogs or pharmaceuticallyacceptable salts thereof, the analog contains fewer amino acids thanfull-length PTH or PTHrP. Preferably the analog contains between about30 and about 40 amino acids. More preferably, the analog contains about34 amino acids.

The invention in another aspect includes pharmaceutical preparationsthat include any of the foregoing analogs or pharmaceutically acceptablesalts thereof, and a pharmaceutically-acceptable carrier.

In another aspect of the invention, methods for treating osteoporosis,promoting bone formation, or inhibiting bone loss in a patient in needof such treatment are provided. The methods include administering aneffective amount of the foregoing analogs, pharmaceutically acceptablesalts thereof, or pharmaceutical preparations to the patient to treatthe osteoporosis, to promote bone formation, or to inhibit bone loss,respectively.

Preferred analogs for use in the methods include bovine PTH analogsincluding an amino acid sequence as set forth in any of SEQ ID NOs:4,13-41, human PTH analogs including an amino acid sequence as set forthin SEQ ID NOs: 5-7 or 42, and human PTHrP analogs including an aminoacid sequence as set forth in SEQ ID NOs: 8-12 or 43.

In certain embodiments, the foregoing methods also include administeringa pharmaceutical agent which is bone resorption inhibiting agent or boneformation promotion agent to the patient. Preferred bone resorptioninhibiting agents or bone formation promotion agents include SelectiveEstrogen-Receptor Modulators (SERMs), estrogens, androgens, cathepsin Kinhibitors, calcium supplements, vitamin D (and metabolites and analogsthereof), thiazide diuretics, calcitonin (and analogs thereof),bisphosphonates, fluoride, integrin antagonists, parathyroid hormone,calcilytics, calcimimetics, inhibitors of Src tyrosine kinase and SrcSH2 inhibitors.

In other embodiments of the foregoing methods, the analog isadministered to the patient at a dosage ranging from 0.1 to 1000micrograms; preferably the dosage ranges from 20 to 200 micrograms.

In still other embodiments of the foregoing methods, the analog isadministered to the patient daily, every second day, every third day,twice per week, every fourth day, every fifth day, every sixth day, oronce per week.

Use of the foregoing analogs in the preparation and/or manufacture of amedicament, particularly a medicament for the treatment of osteoporosis,for the promotion of bone formation or for the inhibition of bone loss.

These and other embodiments of the invention are described in greaterdetail below.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this application, the name of an amino acid may be followedby a superscript number. The superscript number refers to the positionof the amino acid in the sequence. For example, Glu22 means there is aglutamic acid at position 22. All polypeptide sequences mentioned hereinare written according to the generally accepted convention wherein theN-terminal amino acid is on the left and the C-terminal amino acid is onthe right. Numbering starts at the N-terminus and proceeds toward theC-terminus. Accordingly, [Leu^(23,28,31)]hPTHrP means a polypeptidehaving a sequence of hPTHrP in which the wild type residues Phe²³,Ile²⁸, and Ile³¹ have each been replaced with leucine.

As used herein, “hPTH(1-34)” refers to a shortened human sequence of PTH(“hPTH”) having amino acids 1 to 34, as set forth in SEQ ID NO:1.Similarly, “bPTH(1-34)” refers to the bovine sequence of PTH (“bPTH”)having amino acids 1 to 34, as set forth in SEQ ID NO:2. “hPTHrP(1-34)”refers to a shortened form of human parathyroid hormone related protein(“hPTHrP”) having amino acids 1 to 34, as set forth in SEQ ID NO:3.

Preferred polypeptides of the invention include various shortened formsof PTH and PTHrP having amino acid substitutions, i.e., analogs thatcontain fewer amino acids than full-length PTH or PTHrP. The length ofthe analogs can be any length that retains activity of PTH or PTHrP,such as binding to a PTH or PTHrP receptor. The shortened analogs can betested for activity using any of the methods known in the art, some ofwhich are disclosed herein.

The length of the PTH or PTHrP analog can be shortened relative tofull-length PTH or PTHrP by 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41 ,42, 43, 44, 45, 46, 47, 48 ,49, 50or more amino acids. Preferably the PTH or PTHrP analog contains betweenabout 30 and about 40 amino acids, inclusive (30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40). Most preferably, the PTH or PTHrP analog containsabout 34 amino acids.

The PTH or PTHrP analogs of the invention can be based on PTH or PTHrPsequences of any species, although it is preferred that human PTH orPTHrP or bovine PTH or PTHrP serve as the basis for the analog.

The polypeptides of the invention also encompass analogs that have amodified carboxy terminus. Preferred modified analogs include amidatedpolypeptides, in which the terminal carboxy group (—COOH) is convertedto a carboxamide group (—C(O)NH₂). The polypeptides of the inventionalso can be modified with an alkylamide group.

Therefore, “hPTH(1-34)NH₂,” as used herein, refers to a truncated formof human parathyroid hormone having a carboxamide group at the carboxyterminus. “bPTH(1-34)NH₂,” as used herein, refers to a truncated form ofbovine parathyroid hormone having a carboxamide group at the carboxyterminus. Similarly, “hPTHrP(1-34)NH₂,” as used herein, refers to atruncated form of human parathyroid hormone related protein having acarboxamide group at the carboxy terminus.

As used herein, “Ψ(CH₂NH)” indicates a reduced peptide bond between tworesidues. For example, SEQ ID NO:4 has a reduced peptide bond betweenAla¹ and Val² . As used herein, the amino acid “Nle” is norleucine,“Aib” is 2-aminoisobutyric acid, “2-Nal” is 2-naphthyl-alanine, “Thi” is2-thienyl-alanine, “,8-Ala” is beta-alanine, “β-hLeu” isbeta-homo-leucine, “β-hAsp” is beta-aspartic acid, “β-hSer” isbeta-homo-serine, “β-hGlu” is beta-homo-glutamic acid, “β-hThr” isbeta-homo-threonine, “β-hArg” is beta-homo-arginine, “β-hLys” isbeta-homo-lysine, “β-hDap” is beta-homo-diaminopropionic acid(3,4-diaminobutyric acid), “β-hDbu” is beta-homo-diaminobutyric acid(3,5-diaminopentanoic acid), “β-hOrn” is beta-homo-ornithine., “β-hAla”is beta-homo-alanine, “β-hAsn” is beta-homo-asparagine, and “β-hGln” isbeta-homo-glutamine. Thus the invention, in certain aspects, relatespreferably to β³-residues (β-substituted β-amino acid residues) but doesnot exclude β²-residues (α-substituted αamino acid residues). All chiralamino acid residues are of the S-configuration.

In some aspects of the invention, substitutions are made at positions16-20 of PTH or PTHrP, preferably at positions 17-19. As examples ofthis, provided below in Table 1 are analogs of bPTH that are substitutedat the indicated amino acids. The wild-type bPTH amino acid sequence,SEQ ID NO:2, is provided for comparison. Similar substitutions can bemade to hPTH.(SEQ ID NO:1) and to hPTHrP (SEQ ID NO:3), which sequencesalso are listed for comparison.

Thus β-amino acid-containing analogs can have the following structuralpermutations, alone or in combination, and optionally with further aminoacid substitutions as described elsewhere herein:

-   -   Position 16: β-hAsn, β-hSer, β-hGln, β-hThr, β-hAla, β-Ala,        β-hRaa, wherein β-hRaa=NH—CH(R)CH₂CO, where R=Et, nPr,        (CH₂)_(n)CONH₂(n=0, 3 and 4).    -   Position 17: β-hAsp, β-hSer, β-hGlu, β-hThr, β-hAla, β-Ala,        β-hRaa, wherein β-hRaa=NH—CH(R)CH₂CO, where R=Et, nPr,        (CH₂)_(n)CO₂H (n=0, 3 and 4).    -   Position 18: β-hMet, β-hNle, β-hLeu, β-hIle, βhVal, β-hAla,        β-Ala, β-hRaa, wherein β-hRaa=NH—CH(R)CH₂CO, where R=Et, nPr.    -   Position 19: β-hAsp, β-hSer, β-hGlu, β-hThr, β-hArg, β-hLys,        β-hDap, β-hDbu, β-hOrn, β-hAla, β-Ala, β-hRaa, wherein        β-hRaa=NH—CH(R)CH₂CO, where R=(CH₂)_(n)Guanidino or guanidine        group surrogates (n=1, 2 and 4), (CH₂)_(n)CO₂H (n=0, 3 and 4).    -   Position 20: β-hArg, β-hLys, β-hDap, β-hOrn, β-hAla, β-Ala,        β-hRaa, wherein β-hRaa=NH—CH(R)CH₂CO where R=(CH₂)_(n)Guanidino        or guanidine group surrogates (n=1, 2 and 4).

Preferred substitutions include βamino acids β-Ala at position 16, β-Alaor β-hSer at position 17, β-Ala or β-hLeu at position 18, β-Ala orβ-hGlu at position 19, and β-Ala at position 20, as shown in theexamplary analogs presented in Table 1. Norleucine (Nle) is preferablysubstituted for the methionine (Met) at position 18 in those analogswhere there is not a βamino acid at position 18. Guanidine groupsurrogates include the following molecules:

TAβLE 1 Exemplary amino acid substitutions of bPTH SEQ IDPTH/PTHrP/Analog Amino Acid Sequence NOSVSEIQLMHNLGKHLNSMIERVEWLRKKLQDVHNF 1 (hPTH)AVSEIQFMHNLGKHLSSMERVEWLRKKLQDVHNF 2 (bPTH)AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTA 3 (hPTHrP)AVSEIQFMHNLGKHL-β-Ala¹⁶-Ser-Nle-Glu-RVEWLRKKLQDVHNF 18AVSEIQFMHNLGKHLS-Ser-Nle-Glu-β-Ala²⁰-VEWLRKKLQDVHNF 19AVSEIQFMHNLGKHLS-β-Ala¹⁷-Nle-Glu-RVEWLRKKLQDVHNF 20AVSEIQFMHNLGKHLS-Ser-β-Ala¹⁸-Glu-RVEWLRKKLQDVHNF 21AVSEIQFMHNLGKHLS-Ser-Nle-β-Ala¹⁹-RVEWLRKKLQDVHNF 22AVSEIQFMHNLGKHLS-β-hSer¹⁷-Nle-Glu-RVβWLRKKLQDVKNF 23AVSEIQFMHNLGKHLS-Ser-β-hLeu¹⁸-Glu-RVEWLRKKLQDVHNF 24AVSEIQFMHNLGKHLS-Ser-Nle-β-hGlu¹⁹-RVEWLRKKLQDVHNF 25AVSEIQFMHNLGKHLS-β-Ala¹⁷-β-Ala¹⁸-Glu-RVEWLRKKLQDVHNF 26AVSEIQFMHNLGKHLS-Ser-β-Ala¹⁸-β-Ala¹⁹-RVEWLRKKLQDVHNF 27AVSEIQFMHNLGKHLS-β-Ala¹⁷-Nle-β-Ala¹⁹-RVEWLRKKLQDVHNF 28AVSEIQFMHNLGKHLS-β-Ala¹⁷-β-hLeu¹⁸-Glu-RVEWLRKKLQDVHNF 29AVSEIQFMHNLGKHLS-Ser-β-hLeu¹⁸-β-Ala¹⁹-RVEWLRKKLQDVENF 30AVSELQFMHNLGKHLS-Ser-β-Ala¹⁸-β-hGlu¹⁹-RVEWLRKKLQDVHNF 31AVSEIQFMHNLGKHLS-β-Ala¹⁷-Nle-β-hGlu¹⁹-RVEWLRKKLQDVHNF 32AVSEIQFMHNLGKHLS-β-hSer¹⁷-Nle-β-Ala¹⁹-RVEWLRKKLQDVHNF 33AVSEIQFMHNLGKHLS-β-hSer¹⁷-Nle-β-Ala¹⁹-Glu-RVEWLRKKLQDVHNF 34AVSEIQFMHNLGKHLS-β-hSer¹⁷-β-Ala¹⁸-β-Ala¹⁹-RVEWLRKKLQDVHNF 35AVSEIQFMHNLGKHLS-β-Ala¹⁷-β-Ala¹⁸-β-hGlu¹⁹-RVEWLRKKLQDVHNF 36AVSEIQFMHNLGKHLS-β-Ala¹⁷-β-hLeu¹⁸-β-Ala¹⁹-RVEWLRKKLQDVHNF 37AVSEIQFMHNLGKHLS-β-hSer¹⁷-β-hLeu¹⁸-Glu-RVEWLRKKLQDVHNF 38AVSEIQFMHNLGKHLS-Ser-β-hLeu¹⁸-β-hGlu¹⁹-RVEWLRKKLQDVHNF 39AVSEIQFMHNLGKHLS-β-hSer¹⁷-Nle-β-hGlu¹⁹-RVEWLRKKLQDVHNF 40AVSEIQFMHNLGKHLS-β-hSer¹⁷-β-hLeu-β-hGlu¹⁹-RVEWLRKKLQDVHNF 41

As described in the examples below, one set of preferred analogs ofh/bPTH and hPTHrP include the following analogs: TABLE 2 ExemplaryPTH/PTHrP Analogs SEQ ID NO: ANALOG AMINO ACID SEQUENCE 5[Glu^(25,29),Leu^(23,27,31),Lys³⁰]hPTH(1-34)NH₂ 6[Glu^(25,29),Lys³⁰]hPTH(1-34)NH₂ 7 [Leu^(23,27,31)]hPTH(1-34)NH₂ 8[Aib²⁶]hPTHrP(1-34)NH₂ 9[Glu^(22,25,29),Leu^(23,28,31),Lys^(26,30),Thi³²]hPTHrP(1-34)NH₂ 10[Glu^(22,25,29),Leu^(23,28,30,31),Lys²⁶]hPTHrP(1-34)NH₂ 11[Leu^(23,28,31)]hPTHrP(1-34)NH₂ 12[Glu^(22,25,29),Lys^(26,30)]hPTHrP(1-34)NH₂ 13[Nle⁸,β-Ala¹⁸,Nal²³,Tyr³⁴]bPTH(1-34)NH₂ 14[Nle⁸,β-Ala^(18,19),Nal²³,Tyr³⁴]bPTH(1-34)NH₂ 15[Nle^(8,18),β-Ala¹⁹,Nal²³,Tyr³⁴]bPTH(1-34)NH₂ 16[Nle⁸,β-hLeu¹⁸,β-Ala¹⁹,Nal²³,Tyr³⁴]bPTH(1-34)NH₂ 17[Nle⁸,β-Ala^(17,19),β-hLeu¹⁸,Nal²³,Tyr³⁴]bPTH(1-34)NH₂

Additional examples of structural permutations in analogs of h/bPTH andhPTHrP include analogs having one or more pseudopeptide units. Preferredpseudopeptide units include pseudodipeptide units, pseudotripeptideunits, pseudotetrapeptide units, etc., which can be located at anyseries of amino acids in the h/bPTH and hPTHrP analogs. In somepreferred embodiments, the pseudodipeptide unit Xxx¹Ψ(CH₂NH)Yyy² can beone of the following: Xxx=Ala, Gly, Thr, Ser and Yyy=Val, Leu, Me, Ile,Phe. In certain embodiments, analogs containing a pseudodipeptide unitalso contain a 2-aminoisobutyric acid residue substitution at position12 (Aib¹²). Exemplary analogs containing a pseudodipeptide unit includethe following:

[Ala¹Ψ(CH₂NH)Val², Nle^(8,18), Aib¹²,2-Nal²³, Tyr³⁴]bPTH(1-34)NH₂ (SEQID NO:4)

[Ser¹Ψ(CH₂NH)Val²,Nle^(8,18),Aib¹²,2-Nal²³,Tyr³⁴]hpTH(1-34)NH₂ (SEQ IDNO:42)

[Ser¹Ψ(CH₂NH)Val²,Aib¹²]hpTHrP(1-34)NH₂ (SEQ ID NO:43).

In further embodiments, hPTH, bPTH and hPTHrP analogs contain additionalor different amino acid substitutions. For example, instead of thesubstituted Glu residues mentioned above, one can introduce Asp, orNHCH(R)CO, where R is (CH₂)_(n)CO₂H and n=0, 3 and 4. Instead of thesubstituted Leu residues mentioned above one can introduce Nle, Ile,Val, Ala, Met, or NHCH(R)CO, where R is Et, or nBu. Instead of thesubstituted Lys residues mentioned above one can introduce Dap, Dbu, orNHCH(R)CO where R is (CH₂)_(n)NH₂ and n=5-7.

In still other embodiments, the analogs described herein also caninclude an amino acid substitution at position 5, preferably a His orIle substitution. Such substitutions are known to influence binding ofPTH and PTHrP to their receptors (see, e.g., Behar et al., Endocrinology137:4217-4224, 1996). Exchanging between Ile and His in position 5 ofPTH and PTHrP, respectively, modifies sub-type receptor specificity.His⁵-PTH becomes less favorable at the PTH2-Rc compared to parent PTHand Ile⁵-PTHrP becomes more favorable at the PTH1-Rc compared to theparent PTHrP. Preferred PTH/PTHrP analogs targeted for bone anabolictreatment have increased selectivity toward the PTH1-Rc located at boneand kidney, the target tissues for PTH. Hence, substitution ofPTH-derived analogs with His at position 5 is anticipated to increasefavorably their affinity and avidity to the PTH1-Rc.

The polypeptides of the invention have several uses, including, but notlimited to, treating osteoporosis, promoting bone formation, andinhibiting bone loss. Additionally, the polypeptides of the inventionmay be used to treat glucocorticoid-induced bone loss, hypercortisolism(both subclinical and clinical), cancer, hypercalcemia, renal failure orother kidney disorders, renal transplant and accompanyingpharmacological treatments, cholestatic liver diseases, viral hepatitis,liver disorders caused by liver transplant, hyperparathyroid disease,bronchial asthma (including hormone-dependent), disorders due tohaemodialysis, and osteomalacia. In such settings, the polypeptides ofthe invention are administered to patients in need of such treatments ineffective amounts.

Patients include men, women, children, young adults, adults, and elderlyadults. An important group of patients includes post-menopausal women.

The polypeptides of the invention are administered in effective amounts.An effective amount means that amount necessary to delay the onset of,inhibit the progression of, halt altogether the onset or progression of,or diagnose the particular condition being treated. When administered toa subject, effective amounts will depend on the particular conditionbeing treated, the severity of the condition, individual patientparameters including age, sex, physical condition, size and weight,concurrent treatment, frequency of treatment, and the mode ofadministration. These factors are well known to those of ordinary skillin the art and can be addressed with no more than routineexperimentation. It is preferred generally that a maximum dose be used,that is, the highest safe dose according to some medical judgment.

As used herein, “treating osteoporosis” means that the patient's bonedensity increases, remains the same, or does not decrease as rapidly asit would without the administration of a polypeptide of the invention.Generally, it is preferable that the patient's bone density remains thesame or increases. More preferably, the patient's bone density increasesby about 1%, 5%, 10%, or more.

As used herein, “promoting bone formation” means that bone densityincreases by about 1%, 5%, 10%, or more.

In particularly preferred embodiments of the foregoing methods, bonedensity will increase by 5-10% in 12-18 months of treatment.

As used herein, “inhibiting bone loss” means that the patient's bonedensity remains the same, or does not decrease as rapidly as it wouldwithout the compounds of the invention. Preferably, the patient's bonedensity decrease is inhibited by about 85%, 90%, 95%, 99%, or more.

When determining the rate of change or amount of change in bone density,patients may serve as their own controls, or their bone densities may becompared to statistically- derived levels, determined, for example, byclinical trials. One such trial is described by Neer et al. (N. Engl. J.Med. 344:1434-1441, 2001).

Bone density is easily measured using routine methods known to those ofskill in the art. For example, see Neer et al. (N. Engl. J. Med.344:1434-1441, 2001), which provides exemplary methods for analysis theeffect of the administration of PTH and/or PTHrP analogs. Other methodsfor measuring bone density will be known to one of ordinary skill in theart.

When administered, pharmaceutical preparations of the invention areapplied in pharmaceutically acceptable amounts and in pharmaceuticallyacceptable compositions. Such preparations may routinely contain salts,buffering agents, preservatives, compatible carriers, and optionallyother therapeutic ingredients. When used in medicine the salts should bepharmaceutically acceptable, but non-pharmaceutically acceptable saltsmay conveniently be used to prepare pharmaceutically acceptable saltsthereof and are not excluded from the scope of the invention. Suchpharmacologically and pharmaceutically acceptable salts include, but arenot limited to, those prepared from the following acids: hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic,p-toluenesulfonic, tartaric, citric, methanesulfonic, formic, succinic,naphthalene-2-sulfonic, pamoic, 3-hydroxy-naphthalenecarboxylic, andbenzene sulfonic. Also, pharmaceutically acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,ammonium, magnesium, potassium or calcium salts of the carboxylic acidgroup.

Suitable buffering agents include: acetic acid and salts thereof (1-2%W/V); citric acid and salts thereof (1-3% W/V); boric acid and saltsthereof (0.5-2.5% W/V); and phosphoric acid and salts thereof (0.8-2%W/V).

Suitable preservatives include benzalkonium chloride (0.003-0.03% W/V);chlorobutanol (0.3-0.9% W/V); parabens (0.01-0.25% W/V) and thimerosal(0.004-0.02% W/V).

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular combination oftherapeutic agents selected, the severity of the condition or disorderbeing treated, or prevented, the condition of the patient, and thedosage required for therapeutic efficacy. The methods of this invention,generally speaking, may be practiced using any mode of administrationthat is medically acceptable, meaning any mode that produces effectivelevels of the active compounds without causing clinically unacceptableadverse effects. Such modes of administration include oral, rectal,topical, transdermal, sublingual or intramuscular, infusion, parenteral,intravenous, intramuscular, intracavity, as a feed additive, as anaerosol, buccal, aural (e.g., via eardrops), intranasal, inhalation, orsubcutaneous. Direct injection could also be preferred for localdelivery to the site of injury.

Although at present subcutaneous administration is routinely employed inthe administration of PTH and/or PTHrP, oral administration may bepreferred for treatment because of the convenience of the subject(patient) as well as the dosing schedule. Generally, daily oral doses ofactive compounds will be from about 0. 1 microgram per day to 1000micrograms per day. It is expected that oral doses in the range of 0.5to 50 micrograms, in one or several administrations per day, will yieldthe desired results. Dosage may be adjusted appropriately to achievedesired drug levels, local or systemic, depending upon the mode ofadministration. For example, it is expected that intravenousadministration would be from an order to several orders of magnitudelower dose per day compared to the oral doses. In the event that theresponse in a subject is insufficient at such doses, even higher doses(or effective higher doses by a different, more localized deliveryroute) may be employed to the extent that patient tolerance permits.

Preferably the polypeptides of the invention are administeredintermittently, which is known in the art to promote anabolic efficacyof PTH, PTHrP and its analogs. Preferred intermittent administrationschedules include daily, every second day, every third day, twice perweek, every fourth day, every fifth day, every sixth day, and once perweek.

The compositions may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the compounds of the inventioninto association with a carrier which constitutes one or more accessoryingredients. In general, the compositions are prepared by uniformly andintimately bringing the compounds of the invention into association witha liquid carrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the compounds of the inventionis preferably isotonic with the blood of the recipient. This aqueouspreparation may be formulated according to known methods using thosesuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable dilutant or solvent,for example as a solution in 1,3-butane diol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono ordi-glycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables. Carrier formulations suitable for oral,subcutaneous, intravenous, intramuscular, etc. are well known in theart.

Compositions suitable for oral administration may be presented asdiscrete units such as capsules, cachets, tablets, syrups, elixirs orlozenges, each containing a predetermined amount of the compounds of theinvention. Compositions suitable for any pulmonary delivery typicallyare formulated and/or are contained in a nebulizer.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the compounds of the invention, increasingconvenience to the subject and the physician, yet are constructed toprovide the anabolic benefit of the polypeptides of the invention. Manytypes of release delivery systems are available and known to those ofordinary skill in the art. They include polymer based systems such aspolylactic and polyglycolic acid, polyanhydrides and polycaprolactone,nonpolymer systems that are lipids including sterols such ascholesterol, liposomes; phoshpholipids; hydrogel release systems;silastic systems; peptide based system; implants and the like. Specificexamples include, but are not limited to: (a) erosional systems in whichthe polypeptide is contained in a form within a matrix, found in U.S.Pat. Nos. 4,452,775, 4,675,189, and 5,736,152, and (b) diffusionalsystems in-which an active component permeates at a controlled rate froma polymer such as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and5,407,686. In addition, pump-based delivery systems can be used, some ofwhich are adapted for implantation.

Use of a long-term sustained release implant may be particularlysuitable for treatment of chronic conditions.

“Long-term” release, as used herein, means that the implant isconstructed and arranged to deliver therapeutic levels of the activeingredient for at least 7 days, preferably for 30-60 days and morepreferably for longer periods of time (e.g., 12 months or longer). Theimplant may be positioned at a site of injury, but need not be.Long-term sustained release implants are well-known to those of ordinaryskill in the art and include some of the release systems describedabove. One such implant system is described in U.S. Pat. No. 6,159,490.

The polypeptides of the invention may be delivered with other agents fortreating osteoporosis, agents for promoting bone formation, and/oragents for inhibiting bone loss or individually, yet close enough intime to have a synergistic effect on the treatment.

EXAMPLES Example 1 Synthesis of the Polypeptides of the Invention

Peptides were synthesized by the solid-phase methodology (Merrifield, R.B. J. Am. Chem. Soc. 1963, 85, 2149-2154) on an Applied Biosystems 430Apeptide synthesizer using Boc/HOBt/NMP chemistry andp-methylbenzhydrylamine.HCl resin. General protocols for the synthesis,purification and characterization of peptides were reported elsewhere(Zhou, A. T.; Besalle, R.; Bisello, A.; Nakamoto, C.; Rosenblatt, M.;Suva, L. J.; Chorev, M. Proc. Natl. Acad. Sci. USA 1997, 94, 3644-3649;Bisello, A.; Adams, A.; Mierke, D.; Pellegrini, M.; Rosenblatt, M.;Suva, L.; Chorev, M. J. Biol. Chem. 1998, 273, 22498-22505; Nakamoto,C.; Behar, V.; Chin, K. R.; Adams, A. E.; Suva, L. J.; Rosenblatt, M.;Chorev, M. Biochemistry 1995, 34, 10546-10552; Goldman, M. E.; Chorev,M.; Reagan, J. E.; Nuft, R. F.; Levy, J. J.; Rosenblatt, M.Endocrinology 1988, 123, 1468-1475). Specifically, the synthesis wascarried out on a 0.5-mmol scale. The resin-bound side chain protectedBoc[Tyr³⁴]PTH(25-34) was split into two halves and the stepwisesynthesis continued to generate resin-bound side chain protectedBoc[Nle¹⁸, Nal²³, Tyr³⁴]PTH(14-34). At this point the synthesis resumedwith aliquots of 0.05 mmol of resin-bound fully protected 21-residuepeptide which was carried out to the completion of the full sequence.The protocol included double couplings, followed by capping with Ac₂O,for the following positions: Ile⁵, Gln⁶, Phe⁷, His⁸, Lys¹³, His¹⁴,Leu¹⁵, Xxx¹⁸, Yyy¹⁹, Arg²⁰, Val²¹, Leu²², Arg²¹, Glu²², Nal²³, Leu²⁴ andVal³¹(Xxx and Yyy represent either the native residues, Met and Glu,respectively, or the corresponding beta-amino acid residues). Afterhydrogen fluoride cleavage, the peptides were purified by preparativereversed-phase high performance liquid chromatography (RP-HPLC)employing a Vydac® Protein C18 column (300 Å, 15-20 μm, 57×300 mm,Waters, Milford, Mass.). The elution was carried out employing a lineargradient of 0-15% B for 10 min followed by 15-50% B for 120 min (A=0.1%TFA in water; B=0.1% TFA in acetonitrile) at a flow rate of 70 mL/minand monitored at 220 nm. Purity exceeded 97% as determined by analyticalRP-HPLC. Structural integrity of the peptides was confirmed by aminoacid analysis and electrospray mass spectrometry.

Example 2 Determination of IC₅₀

SaOS-2/B10 cells were maintained in RPMI1640 medium supplemented with10% fetal bovine serum (FBS) and 2 mM glutamine at 37° C. in ahumidified atmosphere of 5% CO₂ in air. The medium was changed everythree to four days, and the cells were subcultured every week bytrypsinization.

SaOS-2/B10 cells were maintained for four days after they had reachedconfluence. The medium was replaced with 5% FBS RPS/1640 medium andincubated for 2 hrs at room temperature with 10×10⁴ cpmmono-¹²⁵I-[Nle^(8,18),Tyr³⁴(3-¹²⁵I)]bPTH(1-34)NH₂ in the presence orabsence of a competing tested polypeptide of the invention. The cellswere washed four times with ice-cold PBS and lysed with 0.1M NaOH, andthe radioactivity associated with the cells was counted in ascintillation counter. Synthesis of the radiolabelled[Nle^(8,18),Tyr³⁴(3-¹²⁵I)]bPTH(1-34)NH₂ was carried out as described inGoldman M. E., et al., Endocrinology, (1988), 123, 1468-1475.

The binding assay was conducted on the polypeptides of the inventionhaving SEQ ID NO:5, 6, 7, 9, 10, 11, and 12. The IC₅₀s (half maximalinhibition of binding ofmono-¹²⁵I-[Nle^(8,18),Tyr³⁴(3-¹²⁵I)]bPTH(1-34)NH₂) for the testedanalogs were calculated and shown in Table 3 below: TABLE 3 IC₅₀ ofcertain-hPTH/hPTHrP analogs Sequence Description IC₅₀ (nM) SEQ ID NO: 5[Glu^(25,29), Leu^(23,27,31), Lys³⁰]hPTH(1-34)NH₂ 216 SEQ ID NO: 6[Glu^(25,29), Lys³⁰]hPTH(1-34)NH₂ 30 SEQ ID NO: 7[Leu^(23,27,31)]hPTH(1-34)NH₂ 41 SEQ ID NO: 9 [Glu^(22,25,29),Leu^(23,28,31), Lys^(26,30), Thi³²]hPTHrP(1-34)NH₂ 90 SEQ ID NO: 10[Glu^(22,25,29), Leu^(23,28,30,31), Lys²⁶]hPTHrP(1-34)NH₂ 70 SEQ ID NO:11 [Leu^(23,28,31)]hPTHrP(1-34)NH₂ 18.3 SEQ ID NO: 12 [Glu^(22,25,29),Lys^(26,30)]hPTHrP(1-34)NH₂ 5.5

Example 3 Determination of Stimulation of Adenylate Cyclase Activity

The adenylate cyclase activity induced by each of the testedpolypeptides of the invention was also measured in SaOS-2/B 10 cells asdescribed previously (Rodain et al., J. Clin. Invest. (1983) 72,1511;Goldman, et al., Endocrinology (1988) 123, 1468). Conflúnt SaOS-2/B10cells in 24 wells plates were incubated with 0.5 μCi [³H]adenine (26.9Ci/mmol, New England Nuclear, Boston, Mass. in fresh medium at 37° C.for 2 hrs. and washed twice with Hank's solution. The cells were treatedwith 1 mM IBMX (isobutylmethylxanthine, Sigma, St. Louis, Mo.) in freshmedium for 15 min. and a tested PTH analog was added to the medium toincubate for 5 min. The reaction was stopped by the addition of 1.2M TCAfollowed by sample neutralization with 4N KOH. Cyclic AMP was isolatedby the two-column chromatographic method (Salomon, et al., Anal.Biochem. (1974) 58, 541). The radioactivity was counted in ascintillation counter (Liquid scintillation counter 2200CA, PACKARD,Downers Grove, Ill.). The EC₅₀s (half maximal stimulation of adenylatecyclase) were calculated for the tested PTH analogs and are shown inTable 4 below: TABLE 4 EC₅₀ of certain hPTH/hPTHrP analogs SequenceDescription EC₅₀(nM) SEQ ID NO: 5 [Glu^(25,29), Leu^(23,27,31),Lys³⁰]hPTH(1-34)NH₂ 24 SEQ ID NO: 6 [Glu^(25,29), Lys³⁰]hPTH(1-34)NH₂25.5 SEQ ID NO: 7 [Leu^(23,27,31)]hPTH(1-34)NH₂ 1.12 SEQ ID NO: 9[Glu^(22,25,29), Leu^(23,28,31), Lys^(26,30),Thi³²]hPTHrP(1-34)NH₂ 10SEQ ID NO: 10 [Glu^(22,25,29), Leu^(23,28,30,31), Lys²⁶]hPTHrP(1-34)NH₂5 SEQ ID NO: 11 [Leu^(23,28,31)]hPTHrP(1-34)NH₂ 0.74 SEQ ID NO: 12[Glu^(22,25,29), Lys^(26,30)]hPTHrP(1-34)NH₂ 0.42

Example 4 Determination of IC₅₀

Human embryonic kidney (HEK293) cells, stably transfected withrecombinant hPTH1-receptor (HEK293/C21 cell line, ˜400,000receptors/cell) (Pines, M., Adams, A. E., Stueckle, S., Bessalle, R.,Rashti-Behar, V., Chorev, M., Rosenblatt, M., and Suva, L. J.Endocrinology 1994, 135, 1713-1716), were maintained in D-MEM mediumsupplemented with 10% fetal bovine serum and 2 nM glutamine at 37° C. ina humidified atmosphere of 95% air/5% CO₂. The medium was changed every3-4 days, and the cells were subcultured every week. Ligand stimulatedadenylyl cyclase activity and radioreceptor competition binding assayswere performed on confluent cultures, 1-3 days after a change of medium.

[Nle^(8,18),Nal²³,Tyr³⁴]bPTH(1-34)-NH₂ was radioiodinated and the crudematerial was purified as previously described (Roubini, E., Duong, L.T., Gibbons, S. W., Leu, C. T., Caulfield, M. P., Chorev, M., andRosenblatt, M. Biochemistry 1992, 31, 4026-4033). Briefly, 67 μg ofpeptide in 50 μL phosphate buffer, pH 7.4, in a borosilicate tube,coated with 10 μg Iodogen® (Pierce Chemical), were treated at roomtemperature with 2 mCi [¹²⁵I]Na for 12 min, followed by dilution with200 μL of 0.1% TFA in water. The pure mono-radioiodinated peptide wasisolated on an analytical RP-HPLC Vydac Protein C18 column (TheSeparation Group, Hesperia, Calif.) employing a linear gradient of36-42% B in A for 30 min (A, 0.1% TFA in water; B, 0.1% TFA inacetonitrile) at a flow rate of 1 mL/min, and monitored at 220 nm.

Cells were plated in 24-well tissue culture dishes (Corning Glass Works,Corning, N.Y.) and grown to subconfluency. The cells were then incubatedfor 2 h at room temperature in fresh PBS supplemented medium (0.25 mL)containing 100,000 cpm (˜0.1 nM) of the mono-radioiodinated ligand125I-PTH(1-34) in the absence or presence of increasing concentrationsof unlabeled competing ligand. After incubation, cells were washed twicewith PBS and lysed with 0.1 M NaOH. Radioactivity in the lysate wasmeasured in a γ-counter (Analytic GammaTrac™ 1193). The IC50s for thetested analogs were calculated and shown in Table 5 below: TABLE 5 IC₅₀of certain bPTH analogs Sequence Description IC₅₀(nM) SEQ ID NO: 13[Nle⁸,β-Ala¹⁸, Nal²³, Tyr³⁴]bPTH(1-34)NH₂ 285 ± 85 SEQ ID NO: 14 [Nle⁸,β-Ala^(18,19), Nal²³, Tyr³⁴]bPTH(1-34)NH₂ >1000 SEQ ID NO: 15[Nle^(8,18), β-Ala¹⁹, Nal²³, Tyr³⁴]bPTH(1-34)NH₂ 59 ± 7 SEQ ID NO: 16[Nle⁸, β-hLeu¹⁸, β-Ala¹⁹, Nal²³, Tyr³⁴]bPTH(1-34)NH₂ 117 ± 19 SEQ ID NO:17 [Nle⁸, β-Ala^(17,19), β-hLeu¹⁸, Nal²³, Tyr³⁴]bPTH(1-34)NH₂ >1000

Example 5 Determination of Stimulation of Adenylate Cyclase Activity

Stimulation of adenylyl cyclase activity by the PTH(1-34) analogues wasassayed in stably transfected HEK293/C21 as described before (Nakamoto,C., Behar, V., Chin, K. R., Adams, A. E.; Suva, L. J.; Rosenblatt, M.;Chorev, M. Biochemistry 1995, 34, 10546-10552.). Shortly, cells weregrown to confluence in 24-well culture dishes. They were then incubatedwith 0.5 μCi [³H]adenine in fresh PBS supplemented medium at 37° C. for2 h and further treated with 1 mM 3-isobutyl-1-methylxantine (IBMX) infresh medium for 15 min at 37° C. This treatment was followed by 5 minincubation with the corresponding analogue. The reaction was terminatedby adding 1.2 M trichloroacetic acid and neutralized with 4 N KOH. cAMPwas isolated by the two-column chromatographic method (Solomon, Y.,Londos, C., and Rodbell, M. A. Anal. Biochem. 1974, 58, 541-548).Radioactivity was measured in a liquid scintillation counter (BecimanLS6000IC liquid scintillation counter, Downers Grove, Ill.). The EC₅₀s(half maximal stimulation of adenylate cyclase) were calculated for thetested PTH analogs and are shown in Table 6 below: TABLE 6 EC₅₀ ofcertain bPTH analogs Sequence Description EC₅₀(nM) SEQ ID NO: 13 [Nle⁸,β-Ala¹⁸, Nal²³, Tyr³⁴]bPTH(1-34)NH₂ 3.90 ± 0.22 SEQ ID NO: 14 [Nle⁸,β-Ala^(18,19), Nal²³, Tyr³⁴]bPTH(1-34)NH₂ 13.82 ± 0.02  SEQ ID NO: 15[Nle^(8,18), β-Ala¹⁹, Nal²³, Tyr³⁴]bPTH(1-34)NH₂ 1.51 ± 0.21 SEQ ID NO:16 [Nle⁸, β-hLeu¹⁸, β-Ala¹⁹, Nal²³, Tyr³⁴]bPTH(1-34)NH₂ 2.73 ± 0.31 SEQID NO: 17 [Nle⁸, β-Ala^(17,19), β-hLeu¹⁸, Nal²³, Tyr³⁴]bPTH(1-34)NH₂9.97 ± 1.03

Example 6 Determination of In Vivo Bone Anabolic Activity

In vivo bone anabolic activities of the polypeptides of the inventionare tested by administering the peptide of a formulation containing thepeptide into intact animals or an experimental animal model ofosteopenia. The ovariectomized rat is an established animal model forosteoporosis (Hori, M., et al., Bone Miner., (1988), 3, 193-199; Geral,et al., J. Bone Miner. Res., (1989) 4, Supp. 1, S303; Liu, C-C, et al.,J. Bone Miner. Res. (1990), 5, 973-982; Mosekilde, L, et al.,Endocrinol., (1991), 129, 421-428; Wronski, T. J., et al, Bone, (1994),15, 51-58; Reviewed in Demster D. W., et al., Endocrine Rev., (1993),14, 690-709).

The bone anabolic effects of the compound are determined following 12 to60 days of treatment by assessing the change in bone mineral density bydual energy x-ray absorptiometry or dry weight of femurs or total ashweight (Hori, H., et al., Bone Miner., (1988), 3, 193-199; Hefti, E., etal., Clin. Sci. (1982), 62, 389-396). Increase in the rate of boneformation and mineralization are assessed using metabolic labels, e.g.,tetracycline (Tam, C. S., et al., Endocriniology, (1982), 110, 506-512).Qualitative and quantitative evaluations of changes intrabecular/cortical bone volume and complexity are determined bystandard histomorphometric analysis (Wronski, T. J., et al., Bone,(1994), 15, 51-58; Tam, C. S., et al., Endocrinology (1982), 110,506-612; Podbesek, R., et al., Endocrinology (1983), 112, 1000-1006) ofbone samples from control (untreated) and treated animals.

Each of the patents, patent applications and references that are recitedin this application are herein incorporated in their entirety byreference. Having described the presently preferred embodiments, and inaccordance with the present invention, it is believed that othermodifications, variations and changes will be suggested to those skilledin the art in view of the teachings set forth herein. It is, therefore,to be understood that all such variations, modifications, and changesare believed to fall within the scope of the present invention asdefined by the appended claims.

1. An analog of PTH or PTHrP comprising one or more amino acidsubstitutions selected from the group consisting of: (a) at position 16:β-hAsn, β-hSer, β-hGln, β-hThr, β-hAla, β-Ala, β-hRaa, whereinβ-hRaa=NH—CH(R)CH₂CO, where R=Et, nPr, (CH₂)_(n)CONH₂ (n=0, 3 and 4);(b) at position 17: β-hAsp, β-hSer, β-hGlu, β-hThr, β-hAla, β-Ala,β-hRaa, wherein β-hRaa=NH—CH(R)CH₂CO, where R=Et, nPr, (CH₂)_(n)CO₂H(n=0, 3 and 4); (c) at position 18: β-hMet, β-hNle, β-hLeu, β-hIle,β-hVal, β-hAla, β-Ala, β-hRaa, wherein β-hRaa=NH—CH(R)CH₂CO, where R=Et,nPr; (d) at position 19: β-hAsp, β-hSer, β-hGlu, β-hThr, β-hArg, β-hLys,β-hDap, β-hDbu, β-hOrn, β-hAla, β-Ala, β-hRaa, whereinβ-hRaa=NH—CH(R)CH₂CO, where R=(CH₂)_(n)Guanidino or guanidine groupsurrogates (n=1, 2 and 4), (CH₂)_(n)CO₂H (n=0, 3 and 4); and (e) atposition 20: β-hArg, β-hLys, β-hDap, β-hOrn, β-hAla, β-Ala, β-hRaa,wherein β-hRaa=NH—CH(R)CH₂CO where R=(CH₂)_(n)Guanidino or guanidinegroup surrogates (n=1, 2 and 4), or a pharmaceutically acceptable saltthereof.
 2. The analog or pharmaceutically acceptable salt thereof ofclaim 1, further comprising one or more of the following amino acidsubstitutions: Glu at position 22; Leu or 2-Nal at position 23; Glu atposition 25; Lys or Aib at position 26; Leu at position 27; Leu atposition 28; Glu at position 29; Lys or Leu at position 30; Leu atposition 31; Thi at position 32; and Tyr at position
 34. 3. The analogor pharmaceutically acceptable salt thereof of claim 1, wherein the oneor more amino acid substitutions are selected from the group consistingof β-Ala at position 16, β-Ala or β-hSer at position 17, β-Ala or β-hLeuat position 18, β-Ala or β-hGlu at position 19, and β-Ala at position20.
 4. The analog or pharmaceutically acceptable salt thereof of claim1, wherein Nle is substituted for the Met at position 18 in thoseanalogs where there is not a β amino acid at position
 18. 5-7.(canceled)
 8. The analog or pharmaceutically acceptable salt thereof ofclaim 1, wherein the analog comprises the amino acid sequence set forthas any of SEQ ID NOs:13-41.
 9. The analog or pharmaceutically acceptablesalt thereof of claim 1, further comprising a pseudopeptide unit. 10.The analog or pharmaceutically acceptable salt thereof of claim 9,wherein the pseudopeptide unit is a pseudodipeptide unit comprisingXxx¹Ψ(CH₂NH)Yyy², wherein Xxx is an amino acid selected from the groupconsisting of Ala, Gly, Thr and Ser, and Yyy is an amino acid selectedfrom the group consisting of Val, Leu, Nle, Ile and Phe.
 11. The analogor pharmaceutically acceptable salt thereof of claim 1, furthercomprising an amino acid substitution at position
 5. 12. The analog orpharmaceutically acceptable salt thereof of claims 11, wherein the aminoacid substitution at position 5 is His or Ile.
 13. The analog orpharmaceutically acceptable salt thereof of claim 1, wherein the carboxyterminus is modified.
 14. The analog or pharmaceutically acceptable saltthereof of claim 13, wherein the carboxy terminus modification is anamide or an alkylamide. 15-17. (canceled)
 18. A pharmaceuticalpreparation comprising the analog or pharmaceutically acceptable saltthereof of claim 1, and a pharmaceutically-acceptable carrier.
 19. Amethod for treating osteoporosis in a patient in need of such treatmentcomprising administering an effective amount of the analog orpharmaceutically acceptable salt thereof of claim 1, to the patient totreat the osteoporosis.
 20. A method for promoting bone formation in apatient in need of such treatment comprising administering an effectiveamount of the analog or pharmaceutically acceptable salt thereof ofclaim 1, to the patient to promote bone formation.
 21. A method forinhibiting bone loss in a patient in need of such treatment comprisingadministering an effective amount of the analog or pharmaceuticallyacceptable salt thereof of claim 1, to the patient to inhibit bone loss.22-27. (canceled)
 28. An analog of PTH or PTHrP or a pharmaceuticallyacceptable salt thereof, comprising an amino acid substitution atposition 12 and a pseudopeptide unit.
 29. The analog or pharmaceuticallyacceptable salt thereof of claim 28, wherein the amino acid substitutedat position 12 is 2-aminoisobutyric acid (Aib).
 30. The analog orpharmaceutically acceptable salt thereof of claim 28, wherein thepseudopeptide unit is a pseudodipeptide unit comprisingXxx¹Ψ(CH₂NH)Yyy², wherein Xxx is an amino acid selected from the groupconsisting of Ala, Gly, Thr and Ser, and Yyy is an amino acid selectedfrom the group consisting of Val, Leu, Nle, Ile and Phe.
 31. The analogor pharmaceutically acceptable salt thereof of claim 28, furthercomprising one or more of the following amino acid substitutions: Nle atposition 8; Nle at position 18; 2-Nal at position 23; and Tyr atposition
 34. 32-34. (canceled)
 35. The analog or a pharmaceuticallyacceptable salt thereof of claim 28, wherein the analog comprises theamino acid sequence set forth as any of SEQ ID NOs:4, 42 or
 43. 36. Theanalog or pharmaceutically acceptable salt thereof of claim 28, whereinthe carboxy terminus is modified.
 37. The analog or pharmaceuticallyacceptable salt thereof of claim 36, wherein the carboxy terminusmodification is an amide or an alkylamide. 38-40. (canceled)
 41. Theanalog or pharmaceutically acceptable salt thereof of claim 28, furthercomprising an amino acid substitution at position
 5. 42. The analog orpharmaceutically acceptable salt thereof of claim 41, wherein the aminoacid substitution at position 5 is His or Ile.
 43. A pharmaceuticalpreparation comprising the analog or pharmaceutically acceptable saltthereof of claim 28, and a pharmaceutically-acceptable carrier.
 44. Amethod for treating osteoporosis in a patient in need of such treatmentcomprising administering an effective amount of the analog orpharmaceutically acceptable salt thereof of claim 28, to the patient totreat the osteoporosis.
 45. A method for promoting bone formation in apatient in need of such treatment comprising administering an effectiveamount of the analog or pharmaceutically acceptable salt thereof ofclaim 28, to the patient to promote bone formation.
 46. A method forinhibiting bone loss in a patient in need of such treatment comprisingadministering an effective amount of the analog or pharmaceuticallyacceptable salt thereof of claim 28, to the patient to inhibit boneloss. 47-54. (canceled)
 55. An analog of PTH or PTHrP orpharmaceutically acceptable salt thereof, comprising a set of amino acidsubstitutions selected from the group consisting of set (a): Glu atpositions 25 and 29, and Lys 26 and/or 30; set (b): Leu at positions 23and 31, and Leu at position 27 or position 28; and set (c): Aib atposition
 26. 56. The analog or pharmaceutically acceptable salt thereofof claim 55, wherein the set of amino acid substitutions is set (a), andfurther comprising one or more of the following amino acidsubstitutions: Glu at position 22, Leu at positions 23, 27, 28, 30 and31, and Thi at position
 32. 57. The analog or pharmaceuticallyacceptable salt thereof of claim 56, wherein the analog comprises theamino acid sequence set forth as any of SEQ ID NOs:5, 6, 9, 10 or 12.58. The analog or pharmaceutically acceptable salt thereof of claim 55,wherein the set of amino acid substitutions is set (b), and wherein theanalog comprises the amino acid sequence set forth as SEQ ID NO:7 or SEQID NO:11.
 59. The analog or pharmaceutically acceptable salt thereof ofclaim 55, wherein the set of amino acid substitutions is set (c), andwherein the analog comprises the amino acid sequence set forth as SEQ IDNO:8.
 60. The analog or pharmaceutically acceptable salt thereof ofclaim 55, wherein the carboxy terminus is modified.
 61. The analog orpharmaceutically acceptable salt thereof of claim 60, wherein thecarboxy terminus modification is an amide or an alkylamide. 62-64.(canceled)
 65. The analog or pharmaceutically acceptable salt thereof ofclaim 55, further comprising an amino acid substitution at position 5.66. The analog or pharmaceutically acceptable salt thereof of claims 65,wherein the amino acid substitution at position 5 is His or Ile.
 67. Apharmaceutical preparation comprising the analog or pharmaceuticallyacceptable salt thereof of claim 55, and a pharmaceutically-acceptablecarrier.
 68. A method for treating osteoporosis in a patient in need ofsuch treatment comprising administering an effective amount of theanalog or pharmaceutically acceptable salt thereof of claim 55, to thepatient to treat the osteoporosis.
 69. A method for promoting boneformation in a patient in need of such treatment comprisingadministering an effective amount of the analog or pharmaceuticallyacceptable salt thereof of claim 55, to the patient to promote boneformation.
 70. A method for inhibiting bone loss in a patient in need ofsuch treatment comprising administering an effective amount of theanalog or pharmaceutically acceptable salt thereof of claim 55, to thepatient to inhibit bone loss. 71-77. (canceled)